Identification and classification of cis-regulatory elements in the amphipod crustacean Parhyale hawaiensis.

Emerging research organisms enable the study of biology that cannot be addressed using classical 'model' organisms. New data resources can accelerate research in such animals. Here, we present new functional genomic resources for the amphipod crustacean Parhyale hawaiensis, facilitating the exploration of gene regulatory evolution using this emerging research organism. We use Omni-ATAC-seq to identify accessible chromatin genome-wide across a broad time course of Parhyale embryonic development. This time course encompasses many major morphological events, including segmentation, body regionalization, gut morphogenesis and limb development. In addition, we use short- and long-read RNA-seq to generate an improved Parhyale genome annotation, enabling deeper classification of identified regulatory elements. We discover differential accessibility, predict nucleosome positioning, infer transcription factor binding, cluster peaks based on accessibility dynamics, classify biological functions and correlate gene expression with accessibility. Using a Minos transposase reporter system, we demonstrate the potential to identify novel regulatory elements using this approach. This work provides a platform for the identification of novel developmental regulatory elements in Parhyale, and offers a framework for performing such experiments in other emerging research organisms.

Distinct gene expression dynamics in developing and regenerating crustacean limbs.

Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

Antenna regeneration as an ecotoxicological endpoint in a marine amphipod: a proof of concept using dimethyl sulfoxide and diflubenzuron.

Regeneration is a widely spread process across the animal kingdom, including many species of marine crustaceans. It is strongly linked to hormonal cycles and, therefore, a great endpoint candidate for toxicology studies. We selected the amphipod Parhyale hawaiensis as test organism, already used in ecotoxicological studies and able to regenerate its body appendages. We are proposing a protocol to use the antenna regeneration as a toxicity endpoint. First, we evaluated differences in time of completion of regeneration in males and females after the amputation of one antenna of 6 months old animals. Then we compared the influence of different testing volumes in the regeneration process (100 and 5 mL). We used as testing substances, dimethyl sulfoxide (DMSO) and diflubenzuron, a chitin synthesis inhibitor. The most suitable protocol consisted of volumes of 5 mL in 12-well microplates, with 1 organism per well, 12 organisms per concentration (1:1 females/males) and test time duration of around 5 weeks. DMSO accelerated regeneration time with a NOEC of 0.06%. Diflubenzuron inhibited the time necessary to its completion with a NOEC of 0.32 µg L-1. We conclude that the Parhyale hawaiensis antenna regeneration protocol proposed here is a potential tool in ecotoxicology, but more studies are required for its validation not only to verify its utility for testing chemicals but also environmental samples.

The natural anthraquinone dye emodin: Eco/genotoxicological characterization for aquatic organisms.

Emodin is an anthraquinone secondary metabolite produced by several species of plants and fungi. Emodin is known for its pharmacological versatility, and, in the textile industry, for its good dyeing properties. However, its use in the textile industry can result in the formation and disposal of large volumes of wastewater. Emodin mutagenicity has been shown in bacteria and in human cells, but little is known about its possible toxic, genotoxic, or mutagenic effects in aquatic organisms. We have evaluated the eco/genotoxicity of emodin to aquatic organisms. Emodin was toxic to Daphnia similis (EC50 = 130 µg L-1) and zebrafish embryos (LC50 = 25 µg L-1). No toxicity was observed for Raphidocelis subcapitata, Ceriodaphnia dubia, or Parhyale hawaiensis. Additional biochemistry/molecular studies are needed to elucidate the toxic/mutagenic pathways of emodin in aquatic organisms. The PNEC value for emodin was 0.025 µg L-1. In addition to mutagenicity in the Salmonella/microsome assay, emodin was mutagenic in the micronucleus assay in the amphipod P. hawaiensis. Among the anthraquinone dyes tested to date, natural or synthetic, emodin was the most toxic to aquatic species.

Behavioral circatidal rhythms require Bmal1 in Parhyale hawaiensis.

Organisms living in the intertidal zone are exposed to a particularly challenging environment. In addition to daily changes in light intensity and seasonal changes in photoperiod and weather patterns, they experience dramatic oscillations in environmental conditions due to the tides. To anticipate tides, and thus optimize their behavior and physiology, animals occupying intertidal ecological niches have acquired circatidal clocks. Although the existence of these clocks has long been known, their underlying molecular components have proven difficult to identify, in large part because of the lack of an intertidal model organism amenable to genetic manipulation. In particular, the relationship between the circatidal and circadian molecular clocks, and the possibility of shared genetic components, has been a long-standing question. Here, we introduce the genetically tractable crustacean Parhyale hawaiensis as a system for the study of circatidal rhythms. First, we show that P. hawaiensis exhibits robust 12.4-h rhythms of locomotion that can be entrained to an artificial tidal regimen and are temperature compensated. Using CRISPR-Cas9 genome editing, we then demonstrate that the core circadian clock gene Bmal1 is required for circatidal rhythms. Our results thus demonstrate that Bmal1 is a molecular link between circatidal and circadian clocks and establish P. hawaiensis as a powerful system to study the molecular mechanisms underlying circatidal rhythms and their entrainment.

Measuring concentrations of a dye in the hemolymph of a marine amphipod: Development of a protocol for exposure assessment.

The increasing pollution of aquatic environments due to old and emerging contaminants requires the development of integrative methods for exposure assessment. Internal concentrations are a reliable way to estimate total exposure of contaminants originated from different routes (water, sediment, and food). We developed a protocol to evaluate the concentration of a dye, C.I. Disperse Red 1, in the hemolymph of Parhyale hawaiensis, a marine amphipod. LOD and LOQ were satisfactory to detect the dye in all hemolymph samples. The concentration detected in the hemolymph varied related to exposure time and dye concentration (0.003 to 0.086 µg mL-1). Polynomial regression model was the best fit. The protocol was reliable to detect and quantify dye exposure in marine amphipods and can be considered for future assessments of estuarine and marine regions under the influence of dye processing plants. The method possibly can be easily adapted to other amphipods and other azo dyes.

Marine amphipods (Parhyale hawaiensis) as an alternative feed for the lined seahorse (Hippocampus erectus, Perri 1810): nutritional value and feeding trial.

Finding new alternatives to traditional live preys such as Artemia and rotifers, which do not always promote optimal fish growth and survival, is required for the successful aquaculture of highly specialized predatory species, including seahorses. The present study assessed the nutritional value of an interesting marine amphipod (Parhyale hawaiensis), and evaluates through a feeding trial its potential use as a natural prey for 10-months lined seahorse, Hippocampus erectus. P. hawaiensis showed high levels of valuable lipids (20.4-26.7% on dry matter basis) and polyunsaturated fatty acids (PUFAs) ( 26.4-41% of total FAs), including the long-chain PUFAs (LC-PUFAs) arachidonic acid (ARA) (2.9-7.7%), eicosapentaenoic acid (EPA) (4.3-6.5%) and docosahexaenoic acid (DHA) (2.1-6.2%). A comparison between wild-captured and cultured amphipods revealed a significant improvement of the amphipod FA profile in terms of DHA%, total omega-3 (n3) FAs and n3/n6 ratio when employing both a conventional amphipod culture based on a commercial shrimp diet, and, to a lesser extent, a large (3,500 L) biofloc system. Seahorses fed with frozen/wild amphipods, either singly or in combination with Artemia enriched with Super Selco® (INVE Aquaculture, Belgium) for 57 days, substantially improved seahorse growth and FA profiles in terms of ARA, EPA and DHA%, including indices associated to marine sources, such as Σn3 and n3/n6, compared to a diet based solely on enriched Artemia. These results support the use of marine amphipods as an alternative food organism for juvenile H. erectus and suggest a potential use for general marine aquaculture.

Delivery methods for CRISPR/Cas9 gene editing in crustaceans.

In this mini-review we provide an up-to-date overview of the delivery methods that have been used for CRISPR/Cas9 genomic editing in crustacean species. With embryonic microinjection as the main workforce for delivering CRISPR/Cas9 reagents, biologists working with crustacean species have to tackle the technical challenges involved in microinjection. We use examples of three crustacean species (the branchiopod Daphnia, amphipod Parhyale hawaiensis, and decapod Exopalaemon carinicauda) to provide a technical guide for embryonic microinjection. Moreover, we outline two potentially useful new techniques for delivering CRISPR/Cas9 components into crustaceans, i.e., Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and electroporation.

Higher silver bioavailability after nanoparticle dietary exposure in marine amphipods.

On release into surface waters, engineered silver nanoparticles (AgNPs) tend to settle to sediments and, consequently, epibenthic fauna will be exposed to them through diet. We established Ag uptake and accumulation profiles over time in the hemolymph of a marine amphipod fed with a formulated feed containing AgNPs or AgCl. Silver bioavailability was higher in organisms exposed to AgNPs, indicating that the nanoparticles pose a higher risk of toxicity compared to similar concentrations of AgCl. Environ Toxicol Chem 2019;38:806-810. © 2019 SETAC.

Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb.

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.

Seeing is believing.

A small transparent crustacean called Parhyale hawaiensis has become a powerful model system for the study of limb and appendage regeneration.

Live imaging reveals the progenitors and cell dynamics of limb regeneration.

Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean Parhyale hawaiensis. By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and morphogenesis of regenerating legs with unprecedented resolution and temporal detail. Using these recordings we are able to track cell lineages, to generate fate maps of the blastema and to identify the progenitors of regenerated epidermis. We find that there are no specialized stem cells for the epidermis. Most epidermal cells in the distal part of the leg stump proliferate, acquire new positional values and contribute to new segments in the regenerating leg.

Codon and Amino Acid Usage Are Shaped by Selection Across Divergent Model Organisms of the Pancrustacea.

In protein-coding genes, synonymous codon usage and amino acid composition correlate to expression in some eukaryotes, and may result from translational selection. Here, we studied large-scale RNA-seq data from three divergent arthropod models, including cricket (Gryllus bimaculatus), milkweed bug (Oncopeltus fasciatus), and the amphipod crustacean Parhyale hawaiensis, and tested for optimization of codon and amino acid usage relative to expression level. We report strong signals of AT3 optimal codons (those favored in highly expressed genes) in G. bimaculatus and O. fasciatus, whereas weaker signs of GC3 optimal codons were found in P. hawaiensis, suggesting selection on codon usage in all three organisms. Further, in G. bimaculatus and O. fasciatus, high expression was associated with lowered frequency of amino acids with large size/complexity (S/C) scores in favor of those with intermediate S/C values; thus, selection may favor smaller amino acids while retaining those of moderate size for protein stability or conformation. In P. hawaiensis, highly transcribed genes had elevated frequency of amino acids with large and small S/C scores, suggesting a complex dynamic in this crustacean. In all species, the highly transcribed genes appeared to favor short proteins, high optimal codon usage, specific amino acids, and were preferentially involved in cell-cycling and protein synthesis. Together, based on examination of 1,680,067, 1,667,783, and 1,326,896 codon sites in G. bimaculatus, O. fasciatus, and P. hawaiensis, respectively, we conclude that translational selection shapes codon and amino acid usage in these three Pancrustacean arthropods.